This project aimed at investigating the applicability of fluorescent proteins as probes in second-harmonic generation (SHG) imaging in order to obtain additional intermolecular structural information from already existing, and working cellular systems expressing fusion constructs of proteins of interest covalently linked to fluorescent proteins. SHG microscopy even has the potential to visualize conformational changes in vivo in certain cases.During this project, the second-order nonlinear optical properties of nine fluorescent proteins, measured by the first hyperpolarizability, ß, were determined by frequency-resolved hyper-Rayleigh scattering at the fundamental wavelength (first harmonic) of 800 nm. The chromophores of the fluorescent proteins were confirmed to exhibit SHG, and may be used for optical techniques based on SHG. The intensity of their response depends not only on the wavelength used, due to resonance enhancement, but also on the size of the conjugated system, forming the chromophore, of the proteins. A stronger response, correlated with a larger ß, is inherently linked to a longer conjugated system, also associated with a red-shift in the absorption and fluorescence spectra. This counts for all measured proteins, as long as the chromophore has no inversion centers, which is not the case for the enhanced yellow fluorescent protein, eYFP.SHG microscopy was successfully performed on cultured cells stained with dyes especially designed for nonlinear imaging. Fluorescent proteins turn out to be weak in comparison to these samples for several reasons. Firstly, the measured ßs are considerably smaller than those of the small organic dyes; secondly, the concentration of the fluorescent proteins by expression in the cells will usually be remarkably lower; and thirdly, the fluorescent proteins are linked to proteins-of-interest by a relatively flexible linker, compromising the level of order in the intermolecular orientation of the proteins, an important condition for SHG.However, this project also indicates possible solutions to these downsides, as the ßs of the most red-shifted proteins are approaching the ßs of the dyes. More red-shifted proteins are already available and can be tested using higher wavelengths, increasing resonance enhancement with the same effort. Also, the effect of non-natural amino acids can be investigated, as well as methods to increase control over the orientation of the chromophores. When these directions are followed, SHG microscopy may prove effective with fluorescent proteins.